1. Field of the Invention
The present invention relates generally to the field of cell biology and the neurology of neurodegenerate diseases. More specifically, the present invention relates to the manipulation of receptors/transporters that are involved in the uptake/release of glutamate into or out of cells.
2. Description of the Related Art
In the central nervous system, extracellular glutamate concentrations ([Glu].sub.o) are maintained at 1-2 .mu.M (Benveniste et al. 1984; Nicholls et al., 1990). This is accomplished by a class of recently cloned Na+-dependent transporters (Storck et al. 1992; Pines et al. 1992; Kanai et al., 1992) expressed by neurons and glial cells. Knock-out studies (Rothstein et al. 1996) suggests that the glial transporter, GLT-1, which has the highest affinity for glutamate (K.sub.m =2 .mu.M), is particularly important in controlling [Glu].sub.o.
Intracellular glutamate concentrations ([Glu].sub.i) in astrocytes can readily reach concentrations of several millimolar (Ottersen, 1989; Levi et al., 1992) and thus provide a significant glutamate source in the CNS. Astrocytic glutamate transport can operate in reverse, leading to the non-vesicular release of glutamate (Szatkowski et al. 1990; Attwell et al. 1993), which may contribute to neurotoxic [Glu].sub.o surges under conditions of energy failure or in conjunction with neurodegenerative diseases (Choi et al., 1990; Ogata et al. 1992). In addition to preventing pathological increases in glutamate, astrocytic glutamate uptake also assures synaptic transmission in the healthy brain. Small increases in the resting [Glu].sub.o (.apprxeq.1-2 .mu.M) would change the activation of many glutamate receptors (Patneau et al., 1990; Forsythe et al., 1990; Izumi et al. 1992; Zorumski et al. 1996) and could have pronounced effect on synaptic transmission.
Modulation of glutamate transporter expression and activity in neurons and glial cells has been studied to some extent (Swanson et al. 1997; Gegelashvili et al., 1997). However, the modulation of astrocytic glutamate transport by [Glu].sub.o is largely unknown. One candidate receptor class that could serve as [Glu].sub.o sensors in neurons is the family of metabotropic glutamate receptors (mGluRs). Eight mGluR subtypes have been cloned (Pin et al., 1995) and can be classified according to their amino acid homology, pharmacological profile and signal transduction cascade. Group I receptors (mGluR1, 5) are linked to the phosphoinositol (PI) signaling pathway (Masu et al. 1991; Kawabata et al. 1996); Group II receptors (mGluR 2, 3) and group III receptors (mGluR 4,6,7,8) mediate their effect through changes in cyclic AMP (cAMP) levels (Casabona et al. 1992; Genazzani et al. 1993; Prezeau et al. 1994; Winder et al., 1995; Kemp et al. 1996).
In addition to neurons, glial cells also express mGluR receptors (Tanabe et al. 1993; Miller et al. 1995; Petralia et al. 1996). Immunohistochemical studies suggest that the major glial mGluR receptor is mGluR3 (Tanabe et al. 1993; Testa et al. 1994; Petralia et al. 1996). However, pharmacological studies showed that glial mGluR receptor activation can lead to IP3 mediated calcium release (Masu et al. 1991; Porter et al., 1995) or changes in cAMP levels (Baba et al. 1993; Winder et al. 1996) suggestive of an involvement of Group I and II receptors, respectively. Functional roles for mGluRs in glial cells are just emerging (Winder et al., 1996). Particularly, it has been proposed that glial mGluRs may participate in the communication between neurons and glial cells (Porter et al., 1996; Winder et al. 1996) or may protect neurons from excitotoxic injury (Nicoletti et al. 1996; Bruno et al. 1997).
Parkinson's disease (PD) is a devastating neurological condition that affects a large and growing segment of the aged population. The disease, which affects primarily individuals over age 40, is severely dehabilitating and poses a tremendous societal burden. Clinically, Parkinson's disease presents with tremor, rigidity and deficits in equilibrium and posture. It is now clear that these symptoms result from dysfunction in the basal ganglia, structures consisting of the corpus striatum, globus pallidus and substantia nigra, that receive their major input from motor cortex on which they project back via the thalamus. The principal neurotransmitter used in the nigrostriatal pathway is dopamine.
The presence of large concentrations of dopamine in the striatum was first recognized in the 1950s, and experiments in which dopamine was depleted with reserpine in rats produced symptoms reminiscent of Parkinson's disease. Consistent with these early observations, postmortem basal ganglia from Parkinson's disease patients are severely devoid of dopamine (Hornykiewicz, 1966), suggesting that the symptoms of Parkinson's disease are caused by a dopamine deficiency of the striatum.
The anatomic pathology of Parkinson's disease is well defined, but the pathogenesis remains an enigma. The loss of dopamine-containing neurons in the substantia nigra and the presence of eosinophilic inclusion bodies in these degenerating neurons are the anatomic hallmark of Parkinson's disease , and it is probable that this defined lesion accounts for all it's symptoms.
In the early 1980s, the study of Parkinson's disease received an interesting twist and lead to the first implication of glial cells in the pathogenesis of Parkinsonism. In an effort to produce a "synthetic heroin" not controlled by the FDA, a chemist in California produced 1-methyl-4-phenyl-1-1,2,3,6-tetrahydropyridine (MPTP) as a contaminant. Users of the MPTP contaminated narcotic, within a week of drug use, developed severe and irreversible parkinsonism (Langston et al. 1983). Subsequent controlled administration of MPTP in primates established that MPTP was an exquisitely selective neurotoxin and an excellent animal model of Parkinson's disease became available. The mechanism of action of MPTP is now well understood, and involves its binding to monoamine oxidase (MAO), which converts MPTP to 1-methyl-4-phenylpyridinium (MPP.sup.+). MPP.sup.+ is avidly and selectively accumulated by the catecholamine uptake system in dopamineric neurons (Snyder et al., 1986). Once taken up it presumably causes cell death by inhibiting mitochondrial oxidation (Nicklas et al. 1985).
Interestingly, the enzyme MOA that converts MPTP to the toxic MPP.sup.+ is almost exclusively located in glial cells, particularly in astrocytes (Levitt et al. 1982), and it could be shown experimentally that MPP.sup.+ is produced by glial monoamine oxidase (Ransom et al. 1987). In many instances, disease-related neuronal loss (as in Parkinson's disease) is a consequence of neurotoxicity, the uncontrolled build-up of amino acids in toxic concentrations (Choi, 1988a). Glutamate toxicity, in particular, has been implicated in the neuronal loss associated with stoke, epilepsy, and Alzheimer's disease (Choi, 1988a; Choi et al., 1990b; Choi, 1988b). In the healthy brain, neurotoxicity is prevented by highly effective uptake mechanisms that transfer the transmitter from the extracellular space predominantly into astroglial cells (Nicholls et al., 1990; Walz, 1989). Such high-affinity uptake has been demonstrated for glutamate, GABA, aspartate and dopamine (Walz, 1989; Hertz, 1979). Once inside the cell, the transmitter is metabolized or converted into an inactive precursor form. For instance, the glial enzyme glutamine synthetase converts glutamate into glutamine, which is released to serve as neuronal precursor for glutamate biosynthesis (Hertz, 1979). Not only does the build-up or supply of amino acids at neurotoxic concentration result in neuronal lesion, but a failure of glial cells to effectively sequester neurotoxins will have equally devastating consequences. It was recently shown that glial cells may not only fail to sequester transmitter, but may in fact release amino-acids by a reversal of the transport systems that normally functions in the unidirectional uptake of transmitter into the glial cytoplasm (Szatkowski et al. 1990). Interestingly, dopaminergic neurons in the striatum express glutamate transmitter receptors, suggesting that they are susceptible to glutamate toxicity as well (Mereu et al. 1991).
Thus, the prior art is deficient in drugs and methodology to regulate the levels of glutamate in the extracellular space surrounding neuronal and glial cells. The present invention fulfills this long-standing need and desire in the art.